Nicotine in therapeutic angiogenesis and vasculogenesis

ABSTRACT

Methods are provided for induction of angiogenesis by administration of nicotine or other nicotine receptor agonist. Induction of angiogenesis by the methods of the invention can be used in therapeutic angiogenesis in, for example, treatment of ischemic syndromes such as coronary or peripheral arterial disease.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.11/286,850, filed Nov. 22, 2005, which is a continuation-in-partapplication of U.S. patent application Ser. No. 10/147,389, filed May15, 2002, now U.S. Pat. No. 7,160,904 which is a divisional applicationof U.S. patent application Ser. No. 09/628,226, filed Jul. 28, 2000, nowU.S. Pat. No. 6,417,205; and claims the benefit of U.S. ProvisionalApplication Serial No. 60/146,233, filed Jul. 28, 1999, whichapplications are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates generally to the field of regulation ofangiogenesis and vasculogenesis, particularly to induction ofangiogenesis to promote growth of new vasculature.

BACKGROUND OF THE INVENTION

Angiogenesis and vasculogenesis are processes involved in the growth ofblood vessels. Angiogenesis is the process by which new blood vesselsare formed from extant capillaries, while vasculogenesis involves thegrowth of vessels deriving from endothelial progenitor cells.Angiogenesis and vasculogenesis, and the factors that regulate theseprocesses, are important in embryonic development, inflammation, andwound healing, and also contribute to pathologic conditions such astumor growth, diabetic retinopathy, rheumatoid arthritis, and chronicinflammatory diseases (see, e.g., U.S. Pat. No. 5,318,957; Yancopouloset al. (1998) Cell 93:661-4; Folkman et al. (1996) Cell 87;1153-5; andHanahan et al. (1996) Cell 86:353-64).

Both angiogenesis and vasculogenesis involve the proliferation ofendothelial cells. Endothelial cells line the walls of blood vessels;capillaries are comprised almost entirely of endothelial cells. Theangiogenic process involves not only increased endothelial cellproliferation, but also comprises a cascade of additional events,including protease secretion by endothelial cells, degradation of thebasement membrane, migration through the surrounding matrix,proliferation, alignment, differentiation into tube-like structures, andsynthesis of a new basement membrane. Vasculogenesis involvesrecruitment and differentiation of mesenchymal cells into angioblasts,which then differentiation into endothelial cells which then from denovo vessels (see, e.g., Folkman et al. (1996) Cell 87:1153-5).

Several angiogenic and/or vasculogenic agents with different propertiesand mechanisms of action are well known in the art. For example, acidicand basic fibroblast growth factor (FGF), transforming growth factoralpha (TGF-α) and beta (TGF-β), tumor necrosis factor (TNF),platelet-derived growth factor (PDGF), vascular endothelial cell growthfactor (VEGF), and angiogenin are potent and well-characterizedangiogenesis-promoting agents. In addition, both nitric oxide andprostaglandin (a prostacyclin agonist) have been shown to be mediatorsof various angiogenic growth factors, such as VEGF and bFGF. However,the therapeutic applicability of some of these compounds, especially assystemic agents, is limited by their potent pleiotropic effects onvarious cell types.

Angiogenesis and vasculogenesis have been the focus of intense interestsince these processes can be exploited to therapeutic advantage.Stimulation of angiogenesis and/or vasculogenesis can aid in the healingof wounds, the vascularizing of skin grafts, and the enhancement ofcollateral circulation where there has been vascular occlusion orstenosis (e.g., to develop a “biobypass” around an obstruction due tocoronary, carotid, or peripheral arterial occlusion disease). There isan intense interest in factors that are well-tolerated by the subject,but that are of high potency in effecting stimulation of angiogenesisand/or vasculogenesis.

Related Art

Villablanca ((1998) “Nicotine stimulates DNA synthesis and proliferationin vascular endothelial cells in vitro,” J. Appl. Physiol. 84:2089-98)studied the effects of nicotine on endothelial DNA synthesis, DNArepair, proliferation, and cytoxicity using cultures of bovine pulmonaryartery endothelial cells in vitro.

The reference Carty et al. ((1996) “Nicotine and cotinine stimulatesecretion of basic fibroblast growth factor and affect expression ofmatrix metalloproteinases in cultured human smooth muscle cells,” J VascSurg 24:927-35) demonstrate that nicotine stimulates vascular smoothmuscle cells to produce fibroblast growth factor, and also upregulatesthe expression of several matrix metalloproteinases. The investigatorspropose that these data demonstrate mechanisms by which smoking maycause atherosclerosis and aneurysms.

The reference by Belluardo et al. ((1998) Acute intermittent nicotinetreatment produces regional increases of basic fibroblast growth factormessenger RNA and protein in the tel-and diencephalon of the rat,”Neuroscience 83:723-40) reported that nicotine stimulates the expressionof fibroblast growth factor-2 in rat brain, which the investigatorspropose may explain the neuroprotective effect of nicotine in the ratbrain.

Moffett et al. ((“Increased tyrosine phosphorylation and novel cis-actinelement mediate activation of the fibroblast growth factor-2 (FGF-2)gene by nicotinic acetylcholine receptor. New mechanism fortrans-synaptic regulation of cellular development and plasticity,” MolBrain Res 55:293-305) report that nicotine stimulates the expression offibroblast growth factor-2 in neural crest-derived adrenalpheochromatocytes utilizing a unique transcriptional pathway thatrequires tyrosine phosphorylation. The authors propose that thesefindings suggest that activation of nicotine receptors may be involvedin neural development.

Cucina et al. ((1999) “Nicotine regulates basic fibroblastic growthfactor and transforming growth factor β₁ production in endothelialcells,” Biochem Biophys Res Commun 257:302-12) report that nicotineincreases the release of bFGF, decreases the release of TGFβ1 fromendothelial cells, and increases endothelial mitogenesis. The authorsconclude that these effects may have a key role in the development andprogression of atherosclerosis.

Volm et al. (1999) “Angiogenesis and cigarette smoking in squamous celllung carcinomas: an immunohistochemical study of 28 cases.” AnticancerRes 19(1A):333-6 reports that angiogenesis in lung tumors is linked to apatient's smoking habits.

Macklin et al. (1998) “Human vascular endothelial cells expressfunctional nicotinic acetylcholine receptors,” J. Pharmacol. Exper.Therap. 287:435-9 reports that endothelical cells express bothfunctional nicotinic (neuronal type) and muscarinic acetylcholinereceptors.

U.S. Pat. Nos. 5,318,957; 5,866,561; and 5,869,037 describe use ofvarious compounds (haptoglobin and estrogen) and methods(adenoviral-mediated gene therapy of adipocytes) to effect angiogenesis.

For recent reviews in the field of angiogenesis and vasculogenesis, see,e.g., Yancopoulos et al. (1998) Cell 93:661-4; Folkman et al. (1996)Cell 87;1153-5; and

Hanahan et al. (1996) Cell 86:353-64.

SUMMARY OF THE INVENTION

The present invention features methods for induction of angiogenesis byadministration of nicotine or other nicotine receptor agonist. Inductionof angiogenesis by the methods of the invention can be used intherapeutic angiogenesis in, for example, treatment of ischemicsyndromes such as coronary or peripheral arterial disease.

One object of the present invention to provide a method of controlling,particularly enhancing, angiogenesis, particularly with limited or noadverse effects.

Another object of the present invention is to provide a method oftreating and preventing diseases and ailments involving angiogenesissuch as myocardial and cerebral infarctions, mesenteric or limbischemia, wounds, and vascular occlusion or stenosis.

Another object of this invention is to provide a method of enhancingangiogenesis to accelerate wound healing, or the vascularization of askin graft, musculocutaneous flap or other surgically transplantedtissue; or to enhance the healing of a surgically created anastomosis.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the methods of the invention and compositions used therein asmore fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the chemical structures for the nicotine receptoragonists nicotine, epibatidine, and ABT-154, and for the nicotinereceptor antagonists hexamethonium and mecamylamine.

FIG. 2 is a graph illustrating the effect of oral nicotine uponfibrovascular growth in an animal model.

FIG. 3 is a graph illustrating the effect of locally administerednicotine upon fibrovascular growth in an animal model using the discangiogenesis system.

FIG. 4 is a graph illustrating comparing the relative angiogenicpotencies of the angiogenic factors Del-1 and bFGF with locally orsystemically administered nicotine.

FIG. 5 is a graph illustrating the relative capillary densities innon-ischemic (control; “non-ischemic”) ischemic limbs in the hindlimbischemia model (“ischemic”).

FIG. 6 is a graph illustrating the effect of intramuscularlyadministered nicotine upon capillary density in an animal model ofischemia.

FIG. 7 is a graph illustrating the effect of intramuscularlyadministered nicotine upon capillary density in an untreated,non-ischemic limb of an animal of FIG. 6.

FIG. 8 is a graph illustrating the effect of various compounds onendothelial cell migration.

DEFINITIONS

The term “nicotine receptor agonist” is meant to encompass nicotine(which is understood from to include nicotine derivatives and likecompounds) and other compounds that substantially specifically bind anicotine receptor and provide a pharmacological effect, e.g., inductionof angiogenesis. “Nicotine receptor agonists” encompassnaturally-occurring compounds (including, but not limited to, smallmolecules, polypeptides, peptides, etc., particularlynaturally-occurring plant alkaloids, and the like), endogenous ligands(e.g., purified from a natural source, recombinantly produced, orsynthetic, and further including derivatives and variants of suchendogenous ligands), and synthetically produced compounds (e.g., smallmolecules, peptides, etc).

The term “nicotine” is intended to mean the naturally occurring alkaloidknown as nicotine, having the chemical nameS-3-(1-methyl-2-pyrrolidinyl)pyridine, which may be isolated andpurified from nature or synthetically produced in any manner. This termis also intended to encompass the commonly occurring salts containingpharmacologically acceptable anions, such as hydrochloride,hydrobromide, hydroiodide, nitrate, sulfate or bisulfate, phosphate oracid phosphate, acetate, lactate, citrate or acid citrate, tartrate orbitartrate, succinate, maleate, fumarate, gluconate, saccharate,benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorate and pamoate salts. Nicotine is a colorless to paleyellow, strongly alkaline, oily, volatile, hygroscopic liquid having amolecular weight of 162.23 and the formula shown in FIG. 1.

Unless specifically indicated otherwise, the term “nicotine” furtherincludes any pharmacologically acceptable derivative or metabolite ofnicotine which exhibits pharmacotherapeutic properties similar tonicotine. Such derivatives, metabolites, and derivatives of metabolitesare known in the art, and include, but are not necessarily limited to,cotinine, norcotinine, nornicotine, nicotine N-oxide, cotinine N-oxide,3-hydroxycotinine and 5-hydroxycotinine or pharmaceutically acceptablesalts thereof. A number of useful derivatives of nicotine are disclosedwithin the Physician's Desk Reference (most recent edition) as well asHarrison's Principles of Internal Medicine.

The term “nicotine receptor” as in “nicotine receptor agonist” is meantto encompass the classic pentameric protein of the nicotine receptor(formed by subunits which are symmetrically arranged around a centralion channel) as well as any protein comprising a nicotine binding sitethat stimulates angiogenesis upon binding to nicotine or other nicotinereceptor agonist (e.g., the muscarinic acetylcholine receptor). Use ofthe term “nicotine receptor” in the phrase “nicotine receptor agonist”is not meant to limit the present invention to a theorized mechanismthrough which nicotine or other nicotine receptor agonists stimulateangiogenesis (e.g., by binding a nicotine receptor), but rather is ameans of describing the types of compounds contemplated by the inventionthat can be used to facilitate stimulation of angiogenesis.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect, e.g., stimulation of angiogenesis and/or vasculogenesis. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment” as used herein covers anytreatment of a disease in a mammal, particularly a human, and includes:(a) preventing a disease or condition (e.g., preventing the loss of askin graft or a re-attached limb due to inadequate vascularization) fromoccurring in a subject who may be predisposed to the disease but has notyet been diagnosed as having it; (b) inhibiting the disease, e.g.,arresting its development; or (c) relieving the disease (e.g., enhancingthe development of a Abio-bypass@ around an obstructed vessel to improveblood flow to an organ). In the context of the present invention,stimulation of angiogenesis and/or vasculogenesis is employed forsubject having a disease or condition amenable to treatment byincreasing vascularity and increasing blood flow.

By “therapeutically effective amount of a nicotine receptor agonist” ismeant an amount of a nicotine receptor agonist effective to facilitate adesired therapeutic effect, e.g., a desired level of angiogenic and/orvasculogenic stimulation. The precise desired therapeutic effect willvary according to the condition to be treated.

By “isolated” is meant that the compound is separated from all or someof the components that accompany it in nature. By “substantially purenicotine receptor agonist” is meant that the nicotine receptor agonisthas been separated from components that accompany it in nature.Typically, a nicotine receptor agonist is substantially pure when it isat least 60%, by weight, free from naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, nicotine receptor agonist. A substantially pure nicotinereceptor agonist can be obtained, for example, by extraction from anatural source (e.g., tobacco), by chemically synthesizing the compound,or by a combination of purification and chemical modification. Puritycan be measured by any appropriate method, e.g., chromatography, massspectroscopy, HPLC analysis, etc.

A nicotine receptor agonist is substantially free of naturallyassociated components when it is separated from those contaminants whichaccompany it in its natural state. Thus, a nicotine receptor agonistwhich is chemically synthesized or produced in a cellular systemdifferent from the cell from which it naturally originates will besubstantially free from its naturally associated components.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anicotine receptor agonist” includes a plurality of such agonists andreference to “the nicotine receptor” includes reference to one or morenicotine receptors and equivalents thereof known to those skilled in theart, and so forth. It is further noted that the claims may be drafted toexclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the surprising discovery that nicotineinduces angiogenesis. The inventors' initial inquiries were based on theclinical observation that smokers often have inadequate collateraldevelopment after coronary or peripheral arterial obstruction, i.e., theinventors suspected that nicotine might play a role in inhibition ofangiogenesis. Accordingly, the inventors began by testing the localeffects of nicotine in the disc angiogenesis system (DAS). Unexpectedly,the inventors discovered that nicotine was as or more potent as anangiogenic agent than any growth factor tested in this system, includingDel1 (Penta et al. (1999) J Biol Chem 274(16):11101-9) and bFGF.Additional studies revealed that the potent angiogenic effects ofnicotine were mediated in part by products of the cyclooxygenasecascade, and in part by the NO synthase pathway. Studies using the discangiogenesis system suggested that nicotine may be useful fortherapeutic angiogenesis. However, because angiogenesis is such acomplex process, to demonstrate proof of principle that an agent hasutility for therapeutic angiogenesis, the agent was tested in an animalmodel of disease that requires angiogenesis for its treatment.Accordingly, studies were performed in an animal model of arterialocclusion (the murine hindlimb ischemia model). Using this model ofarterial occlusion, the inventors obtained compelling evidence thatnicotine induces therapeutic angiogenesis.

Thus, the inventors have discovered that nicotine, a component oftobacco smoke, provides the basis of a new therapeutic approach toenhance angiogenesis in the treatment of coronary, peripheral, or otherocclusive arterial diseases; and for the enhancement of wound healingand the improved vascularization of surgically transplanted tissues ororgans (e.g., skin grafts or reattached limbs). In view of its similaror relatively increased angiogenic potency relative to other,conventional angiogenic agents, nicotine has significant advantage overcurrent candidates as the basis of therapeutic angiogenesis. Moreover,the pharmacology and pharmacokinetics of nicotine have already beenwell-characterized in the context of smoking (e.g., in an effort tofacilitate smoking cessation) and methods for slow release and localdelivery have already been intensively investigated. Processes for themanufacture of nicotine and nicotine agonists are also wellcharacterized. Furthermore, these small molecules are more easilysynthesized and stored than complex angiogenic peptides.

Accordingly, the invention encompasses methods and compositions forstimulation of angiogenesis and/or vasculogenesis by administration of anicotine receptor agonist. Of particular interest is the stimulation ofangiogenesis and/or vasculogenesis in vivo to effect increase in bloodflow, increased capillary density, and/or increased vascularity within,adjacent, or around an ischemic site.

Nicotine and Other Nicotine Receptor Agonists

The methods of the invention to stimulate angiogenesis are accomplishedby administration of a nicotine receptor agonist, particularly nicotine,nicotine metabolite, or nicotine derivative. Methods for production ofnicotine derivatives and analogues are well known in the art. See, e.g.,U.S. Pat. Nos. 4,590,278; 4,321,387; 4,452,984; 4,442,292; and4,332,945.

Additional nicotine receptor agonists of interest include, but are notnecessarily limited to, naturally occurring plant alkaloids (e.g.,lobeline, lobeline derivatives, and the like), which plant-derivedcompounds can be provided in a herbal preparation (e.g., in the form ofdried tobacco leaves, in a poultice, in a botanical preparation, etc.),in isolated form (e.g., separated or partially separated from thematerials that naturally accompany it), or in a substantially purifiedform. Other nicotine receptor agonists include choline esteraseinhibitors (acetylcholinesterase inhibitors) (e.g., inhibitors thatincrease local concentration of acetylcholine), derivatives ofepibatidine that specifically bind the neuronal type of nicotinicreceptors (with reduced binding to the muscarinic receptor) and havingreduced deleterious side-effects (e.g., Epidoxidine, ABT-154, ABT-418,ABT-594; Abbott Laboratories (Damaj et al. (1998) J. Pharmacol Exp.Ther. 284:1058-65, describing several analogs of epibatidine of equalpotency but with high specificity to the neuronal type of nicotinicreceptors). Further nicotine receptor agonists of interest include, butare not necessarily limited to, N-methylcarbarnyl andN-methylthi-O-carbamyl esters of choline (e.g., trimethylarninoethanol)(Abood et al. (1988) Pharmacol. Biochem. Behav. 30:403-8); acetylcholine(an endogenous ligand for the nicotine receptor); and the like.

Suitable acetylcholinesterase inhibitors ARICEPT (donepezil; U.S. Pat.No. 4,895,841); EXELON (rivastigmine ((S)-[N-ethyl-3-[1-(dimethylamino)ethyl]phenyl carbamate); U.S. Pat. No. 5,603,176 andU.S. Pat. No. 4,948,807); metrifonate((2,2,2-trichloro-1-hydroxyethyl)phosphonic acid dimethyl ester; U.S.Pat. Nos. 2,701,225 and 4,950,658); galantamine (U.S. Pat. No.4,663,318); physostigmine (Forest, USA); tacrine(1,2,3,4-tetrahydro-9-acridinamine; U.S. Pat. No. 4,816,456);heptylphysostigmine; neostigmine; eptastigmine; huperzine A(5R-(5α,9β,11Ε))-5-amino-11-ethylidene-5,6,9,10-tetrahydro-7-methyl-5,9-methaneocycloocta(b)pyridin-2-(1H)-one);huperizine A analogs;icopezil(5,7-dihydro-3-(2-(1-(phenylmethyl)-4-piperidinyl)ethyl)-6H-pyrrolo(3,2-f)-1,2-benzisoxazol-6-one;U.S. Pat. No. 5,750,542 and WO 92/17475); edrophonium chloride;citicoline; matrifonate; 7-methoxytacrine; ipidacrine; zifrosilone;anseculin; suronacrine; linopirdine; velnacrine; galanthaminederivative, e.g., as described in U.S. Pat. No. 6,316,439;2-Phenoxy-indan-1-one derivatives; an(1-indanone)-(1,2,3,6-tetrahydropyridine) compound as described in U.S.Patent Publication No. 20050124642; zanapezil (TAK-147); memantine(Ebixa); and an acetylcholinesterase inhibitor comprising a1-benzylpyridinium salt, as described in U.S. Patent Publication No.20030069289. 2-Phenoxy-indan-1-one derivatives that areacetylcholinesterase inhibitors have been described in the literature.See, e.g., Sheng et al. (2005) Bioorg. Med. Chem. Lett. 15:3834-3737.Also suitable for use is a prodrug of an acetylcholinesterase inhibitor,a pharmaceutically acceptable salt or solvate of an acetylcholinesteraseinhibitor, and a pharmaceutically acceptable salt or solvate of aprodrug of an acetylcholinesterase inhibitor. Suitableacetylcholinesterase inhibitors are also found in, e.g., WO 2000/48599;WO 1999/64421; WO 2000/51985; and WO 2001/16105; and Dorronsoro et al.(2003) Expert Opin. Ther. Patents 13:1725.

In some embodiments, the acetylcholinesterase inhibitor is a compound ofFormula I:

In some embodiments, the acetylcholinesterase inhibitor is a compound asdescribed in U.S. Pat. No. 6,277,866. In other embodiments, theacetylcholinesterase inhibitor is a compound as described in U.S. Pat.No. 6,706,741. In other embodiments, the acetylcholinesterase inhibitoris a compound as described in U.S. Pat. No. 6,906,083. In otherembodiments, the acetylcholinesterase inhibitor is a compound asdescribed in U.S. Pat. No. 6,677,330. In other embodiments, theacetylcholinesterase inhibitor is a compound as described in U.S. Pat.No. 6,319,916. In other embodiments, the acetylcholinesterase inhibitoris a compound as described in any of WO 2000/48599; WO 1999/64421; WO2000/51985; and WO 2001/16105.

In some embodiments, the acetylcholinesterase inhibitor is any one ofcompounds 1-12, as shown below:

Also suitable for use is tolserine, where tolserine has the followingstructure (Formula II):

Nicotine receptor agonists can also be readily identified using methodswell known in the art. For example, the ability of a candidate nicotinereceptor agonist can be screened for binding to a nicotine receptor invitro, and the ability of the candidate agent to stimulate angiogenesisand/or vasculogenesis can be assessed in vivo (e.g., using the discangiogenesis system (DAS), in the hindlimb ischemia model, etc.).

Pharmaceutical Compositions

Upon reading the present specification, the ordinarily skilled artisanwill appreciate that the pharmaceutical compositions comprising anicotine receptor agonist described herein can be provided in a widevariety of formulations. More particularly, the nicotine receptoragonist can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid(e.g., gel), liquid or gaseous forms, such as tablets, capsules,powders, granules, ointments, solutions, suppositories, injections,inhalants and aerosols. Where the nicotine receptor agonist is anaturally-occurring compound, the pharmaceutical composition can also beprovided as an herbal preparation (e.g., in the form of tobacco leaves,as a poultice of plant matter, in a botanical preparation, etc.).

The nicotine receptor agonist formulation used will vary according tothe condition or disease to be treated, the route of administration, theamount of nicotine receptor agonist to be administered, and othervariables that will be readily appreciated by the ordinarily skilledartisan. In general, and as discussed in more detail below,administration of nicotine receptor agonists can be either systemic orlocal, and can be achieved in various ways, including, but notnecessarily limited to, administration by a route that is parenteral,intravenous, intra-arterial, inter-pericardial, intramuscular,intraperitoneal, transdermal, transcutaneous, subdermal, intradermal,intrapulnonary, etc.

In pharmaceutical dosage forms, the nicotine receptor agonist may beadministered in the form of their pharmaceutically acceptable salts, orthey may also be used alone or in appropriate association, as well as incombination, with other pharmaceutically active compounds. The followingmethods and excipients are merely exemplary and are in no way limiting.

The nicotine receptor agonist can be formulated into preparations forinjection by dissolving, suspending or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

Formulations suitable for topical, transcutaneous, and transdermaladministration, e.g., to administer the nicotine receptor agonistdirectly to a wound, may be similarly prepared through use ofappropriate suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. Topical formulations may be alsoutilized with a means to provide continuous administration of nicotineor other nicotine receptor agonist by, for example, incorporation intoslow-release pellets or controlled-release patches.

The nicotine receptor agonist can also be formulated in a biocompatiblegel, which gel can be applied topically (e.g., to facilitate woundhealing) or implanted (e.g., to provide for sustained release ofnicotine receptor agonist at an internal treatment site). Suitable gelsand methods for formulating a desired compound for delivery using thegel are well known in the art (see, e.g., U.S. Pat. Nos. 5,801,033;5,827,937; 5,700,848; and MATRIGEL™).

For oral preparations, the nicotine receptor agonist can be used aloneor in combination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The nicotine receptor agonist can be utilized in aerosol formulation tobe administered via inhalation. The compounds of the present inventioncan be formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the nicotine receptor agonist can be made intosuppositories by mixing with a variety of bases such as emulsifyingbases or water-soluble bases. The compounds of the present invention canbe administered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and/or animalsubjects, each unit containing a predetermined quantity of nicotinereceptor agonist calculated in an amount sufficient to produce thedesired angiogenic and/or vasculogenic effect in association with apharmaceutically acceptable diluent, carrier or vehicle. Thespecifications for the unit dosage forms of the present invention dependon the particular compound employed and the effect to be achieved, andthe pharmacodynamics associated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

In addition to one or more nicotine receptor agonists, thepharmaceutical formulations according to the invention can comprise orbe administered in parallel with agents that enhance angiogenesis byenhancing nitric oxide (NO) levels (e.g, by enhancing activity of NOsynthase, by enhancing release of NO, etc.) or prostacyclin levels(e.g., by enhancing activity prostacyclin synthase, by enhancing releaseof prostacyclin, etc.) Exemplary NO level-enhancing agents include, butare not necessarily limited to, L-arginine, L-lysine, and peptidesenriched with these amino acids which can serve as substrates for NO;agents that preserve NO activity such as antioxidants (e.g., tocopherol,ascorbic acid, ubiquinone) or antioxidant enzymes (e.g., superoxidedismutase); and agents which can enhance NO synthase activity (e.g.,tetrahydrobiopterin, or precursors for tetrahydrobiopterin (e.g.,sepiapterin)); and the like. Exemplary prostacyclin level-enhancingagents include, but are not limited to precursors for prostacyclin suchas eicosopentanoic acid and docosohexanoic acid; and prostanoids such asprostaglandin E1 and its analogues; and the like.

Alternatively or in addition, the pharmaceutical compositions accordingto the invention can comprise additional angiogenesis-inducing and/orvasculogenesis-inducing agents that act through pathways other than thenicotine receptor (e.g., vascular endothelial growth factor (VEGF),fibroblast growth factor (FGF) (e.g., acidic FGF (aFGF), basic FGF(bFGF), Del1, etc.).

Particularly where the nicotine receptor agonist is to be delivered forlocal application, e.g, by an intramuscular route, it may be desirableto provide the nicotine receptor agonist in a gel or matrix that cansupport angiogenesis, e.g., migration and proliferation of vascularcells into the matrix with endothelial tube formation. The gel or matrixcan thus provide at least the initial substrate upon which new vesselsform. For example, the gel or matrix can be extruded into an ischemicregion to form a path for new blood vessel formation so as to bypass anobstruction in the area.

Induction of Angiogenesis in vivo

In order to accomplish stimulation of angiogenesis in vivo (e.g., as inthe context of therapeutic angiogenesis), nicotine or other nicotinereceptor agonists can be administered in any suitable manner, preferablywith pharmaceutically acceptable carriers. One skilled in the art willreadily appreciate that the a variety of suitable methods ofadministering nicotine or other nicotine receptor agonist in the contextof the present invention to a subject are available, and, although morethan one route can be used to administer a particular compound, aparticular route can provide a more immediate, more effective, and/orassociated with fewer side effects than another route. In general, anicotine receptor agonist can be administered according to the method ofthe invention by, for example, a parenteral, intravenous,intra-arterial, inter-pericardial, intramuscular, intraperitoneal,transdermal, transcutaneous, subdermal, intradermal, or intrapulmonaryroute.

In order to avoid the side-effects associated with systemic nicotine, itmay be preferable to administer nicotine locally (either alone or withagents to enhance the activity of the NO synthase or prostacyclinsynthase pathways). Local administration can be accomplished by, forexample, direct injection (e.g., intramuscular injection) at the desiredtreatment site, by introduction of the nicotine receptor agonistformulation intravenously at a site near a desired treatment site (e.g.,into a vessel or capillary that feeds a treatment site), byintra-arterial or intra-pericardial introduction, by introduction (e.g.,by injection or other method of implantation) of a nicotine receptoragonist formulation in a biocompatible gel or capsule within or adjacenta treatment site, by injection directly into muscle or other tissue inwhich increased blood flow and/or increased vascularity is desired, byrectal introduction of the formulation (e.g., in the form of asuppository to, for example, facilitate vascularization of a surgicallycreated anastomosis after resection of a piece of the bowel), etc.

In one particular application of interest, the nicotine receptor agonistformulation is employed in a “biobypass” method, wherein instead ofperforming a more invasive procedure, such as a coronary bypassoperation, a nicotine receptor agonist formulation is administered toinduce growth of new blood vessels around the blocked region. In thisembodiment, the nicotine receptor agonist formulation can beadministered in the area of and/or proximate to the ischemic tissue tostimulate angiogenesis.

In some embodiments it may be desirable to deliver the nicotine receptoragonist directly to the wall of a vessel. One exemplary method of vesselwall administration involves the use of a drug delivery catheter,particularly a drug delivery catheter comprising an inflatable balloonthat can facilitate delivery to a vessel wall. Thus, in one embodimentthe method of the invention comprises delivery of a nicotine receptoragonist to a vessel wall by inflating a balloon catheter, wherein theballoon comprises a nicotine receptor agonist formulation covering asubstantial portion of the balloon. The nicotine receptor agonistformulation is held in place against the vessel wall, promotingadsorption through the vessel wall. In one example, the catheter is aperfusion balloon catheter, which allows perfusion of blood through thecatheter while holding the nicotine receptor agonist against the vesselwalls for longer adsorption times. Examples of catheters suitable fornicotine receptor agonist application include drug delivery cathetersdisclosed in U.S. Pat. Nos. 5,558,642; 5,554,119; 5,591,129; and thelike.

In another embodiment of interest, the nicotine receptor agonistformulation is delivered in the form of a biocompatible gel, which canbe implanted (e.g., by injection into or adjacent a treatment site, byextrusion into or adjacent a tissue to be treated, etc.). Gelformulations comprising a nicotine receptor agonist can be designed tofacilitate local release of the nicotine receptor agonist and otheractive agents for a sustained period (e.g., over a period of hours,days, weeks, etc.). The gel can be injected into or near a treatmentsite, e.g., using a needle or other delivery device. In one embodiment,the gel is placed into or on an instrument which is inserted into thetissue and then slowly withdrawn to leave a track of gel, resulting instimulation of angiogenesis along the path made by the instrument. Thislatter method of delivery may be particularly desirable for the purposeof directing course of the biobypass.

In other embodiments it may be desirable to deliver the nicotinereceptor agonist formulation topically, e.g., for localized delivery,e.g., to facilitate wound healing. Topical application can beaccomplished by use of a biocompatible gel, which may be provided in theform of a patch, or by use of a cream, foam, and the like. Several gels,patches, creams, foams, and the like appropriate for application towounds can be modified for delivery of nicotine receptor agonistformulations according to the invention (see, e.g., U.S. Pat. Nos.5,853,749; 5,844,013; 5,804,213; 5,770,229; and the like). In general,topical administration is accomplished using a carrier such as ahydrophilic colloid or other material that provides a moist environment.Alternatively, for the purpose of wound healing the nicotine agonistcould be supplied, with or without other angiogenic agents in a gel orcream that could be applied to the wound. An example of such anapplication would be as a sodium carboxymethylcellulose-based topicalgel with a low bioburden containing the nicotine agonist and otheractive ingredients together with preservatives and stabilizers.

In other embodiments, the nicotine receptor agonist formulation isdelivered locally or systemically, preferably locally, using atransdermal patch. Several transdermal patches are well known in the artfor systemic delivery of nicotine to facilitate smoking cessation, andsuch patches may be modified to provide for delivery of an amount ofnicotine receptor agonist effective to stimulate angiogenesis accordingto the invention (see, e.g., U.S. Pat. Nos. 4,920,989; and 4,943,435,NICOTROL™ patch, and the like).

In other methods of delivery, the nicotine receptor agonist can beadministered using iontophoretic techniques. Methods and compositionsfor use in iontophoresis are well known in the art (see, e.g., U.S. Pat.Nos. 5,415,629; 5,899,876; 5,807,306; and the like).

The desirable extent of angiogenesis will depend on the particularcondition or disease being treated, as well as the stability of thepatient and possible side-effects. In proper doses and with suitableadministration, the present invention provides for a wide range ofdevelopment of blood vessels, e.g., from little development toessentially full development.

Stimulation of angiogenesis and/or vasculogenesis by administration of anicotine receptor agonist can be controlled by administration ofcompounds that interfere with nicotine receptor agonist-mediatedangiogenic stimulation. In this sense, the invention also provides for ameans of controlling or inhibiting angiogenesis by interfering with therole of the nicotine in the angiogenic process. This may beaccomplished, for example, administration of agents that inhibit theability of nicotine to mediate its effects through the nicotine receptor(e.g., by inhibiting binding of nicotine to the nicotine receptor).Exemplary nicotinic receptor antagonists include hexamethonium andmecamylamine (formulas provided in FIG. 1). Alternatively, nicotinereceptor agonist-mediated angiogenesis can be controlled or inhibited byadministration of inhibitors of processes downstream of nicotinereceptor signaling. For example, inhibitors of nitric oxide synthaseand/or prostacyclin antagonists can be administered to inhibitangiogenesis. The angiogenesis inhibitor may be administered in the samemanner and dosages to mammals, such as humans, as described with respectto the nicotine receptor agonist.

Where the subject has had exposure, particularly chronic exposure, to anicotine receptor agonist (e.g., as in a subject who is a smoker and whohas had prior, particularly chronic, systemic exposure to nicotine), thenicotine receptor or other nicotine receptor agonist-binding receptorthat mediates stimulation of angiogenesis may be present at lower levelsthan in subjects who have not had previous exposure or chronic exposureto a nicotine receptor agonist. In such subjects, it thus may bedesirable to administer an initial course of a nicotine receptorantagonist to stimulate an increase in nicotine binding-receptors.

Dose

The dose of nicotine or other nicotine receptor agonist administered toa subject, particularly a human, in the context of the present inventionshould be sufficient to effect a therapeutic angiogenic response in thesubject over a reasonable time frame. The dose will be determined by thepotency of the particular nicotine receptor agonist employed and thecondition of the subject, as well as the body weight of the subject tobe treated. For example, the level or affinity or both of the nicotinereceptor agonist for the nicotine receptor may play a role in regulatingthe compound's angiogenic activity. The size of the dose also will bedetermined by the existence, nature, and extent of any adverseside-effects that might accompany the administration of a particularcompound.

In determining the effective amount of nicotine or nicotine receptoragonist in the stimulation of angiogenesis, the route of administration,the kinetics of the release system (e.g., pill, gel or other matrix),and the potency of the nicotine agonist is considered so as to achievethe desired angiogenic effect with minimal adverse side effects. Thenicotine receptor agonist will typically be administered to the subjectbeing treated for a time period ranging from a day to a few weeks,consistent with the clinical condition of the treated subject.

The following dosages assume that nicotine is being administered, or anicotine receptor agonist with similar potency and efficacy as nicotine.As will be readily apparent to the ordinarily skilled artisan, thedosage is adjusted for nicotine receptor agonists according to theirpotency and/or efficacy relative to nicotine. If given orally or as aninhalant, the dose may be in the range of about 0.01 mg to 10 mg, given1 to 20 times daily, and can be up to a total daily dose of about 0.1 mgto 100 mg. If applied topically, for the purpose of a systemic effect,the patch or cream would be designed to provide for systemic delivery ofa dose in the range of about 0.01 mg to 10 mg. If the purpose of thetopical formulation (e.g., cream) is to provide a local angiogeniceffect, the dose would likely be in the range of about 0.001 mg to 1 mg.If injected for the purpose of a systemic effect, the matrix in whichthe nicotine agonist is administered is designed to provide for asystemic delivery of a dose in the range of about 0.001 mg to 1 mg. Ifinjected for the purpose of a local effect, the matrix is designed torelease locally an amount of nicotine agonist in the range of about0.003 mg to 1 mg.

Regardless of the route of administration, the dose of nicotine receptoragonist can be administered over any appropriate time period, e.g., overthe course of 1 to 24 hours, over one to several days, etc. Furthermore,multiple doses can be administered over a selected time period. Asuitable dose can be administered in suitable subdoses per day,particularly in a prophylactic regimen. The precise treatment level willbe dependent upon the response of the subject being treated. In thetreatment of some individuals with nicotine receptor agonists, it may bedesirable to utilize a “megadosing” regimen. In such a treatment, alarge dose of the nicotine receptor agonist is administered to anindividual, time is allowed for the compound to act, and then a suitablereagent, e.g., a nicotine receptor antagonist, is administered to theindividual to render the active compound ineffective or to reduce itssystemic side-effects.

Conditions Amenable to Treatment by Nicotine Receptor Agonist-MediatedInduction of Angiogenesis

The methods and nicotine receptor agonist-comprising compositions of theinvention can be used to treat a variety of conditions that wouldbenefit from stimulation of angiogenesis, stimulation of vasculogenesis,increased blood flow, and/or increased vascularity.

Examples of conditions and diseases amenable to treatment according tothe method of the invention include any condition associated with anobstruction of a blood vessel, e.g., obstruction of an artery, vein, orof a capillary system. Specific examples of such conditions or diseaseinclude, but are not necessarily limited to, coronary occlusive disease,carotid occlusive disease, arterial occlusive disease, peripheralarterial disease, atherosclerosis, myointimal hyperplasia (e.g., due tovascular surgery or balloon angioplasty or vascular stenting),thromboangiitis obliterans, thrombotic disorders, vasculitis, and thelike. Examples of conditions or diseases that can be prevented using themethods of the invention include, but are not necessarily limited to,heart attack (myocardial infarction) or other vascular death, stroke,death or loss of limbs associated with decreased blood flow, and thelike.

Other forms of therapeutic angiogenesis include, but are not necessarilylimited to, the use of nicotine receptor agonists to accelerate healingof wounds or ulcers; to improve the vascularization of skin grafts orreattached limbs so as to preserve their function and viability; toimprove the healing of surgical anastomoses (e.g., as in re-connectingportions of the bowel after gastrointestinal surgery); and to improvethe growth of skin or hair.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Materials and Methods

The following is a description of the methods and materials used in thespecific examples below.

Animals

Eight to ten week old female wild type C57BL/6J mice were used. The miceweighed 20-25 grams (Jackson Laboratories, Bar Harbor, Me. andDepartment of Comparative Medicine (DCM), Stanford, Calif.), and weremaintained as previously described (Maxwell et al. (1998) Circulation1998 98(4):369-374).

Disc Angiogenesis System (DAS)

To study whether nicotine induces angiogenesis in vivo, we employed thedisc angiogenesis system (DAS) (Kowalski et al. (1992) Exp Mol Pathol56(1):1-19; Fajardo et al. (1998) Lab Invest 58:718-7244).

Preparation of the disc. The DAS consisted of a disc (11 mm in diameterand 1 mm thickness) made of a polyvinyl alcohol sponge (Kanebo PVA,RippeyCo., Santa Clara, Calif.). Both sides were covered withnitrocellulose cell-impermeable filters (Millipore filters, 0.45* m inpore diameter, Millipore, SF, Calif.) of the same diameter as the spongedisc, fixed to the sponge using Millipore glue#1 (xx70000.00,Millipore). As a result, cells (and thus vessels) could penetrate orexit only through the rim of the disc (Kowalski et al. (1992) Exp MolPathol 56(1):1-19; Fajardo et al. (1998) Lab Invest 58:718-7244).

In order to study the local effect of nicotine on angiogenesis, nicotinewas placed in a pellet which was added directly to the disc. Briefly, a1.5-mm core (pellet) was cut from the disc center. Both the pellets anddiscs were sterilized prior to assembly in a laminar flow hood. Thepellet was loaded with up to 20 mcl of the nicotine solution andsubsequently air-dried. We placed in the disc pellets with eithervehicle (PBS, Sigma, Chemical Co., St Louis, Mo., n=5) or nicotine (10-6M, Aldrich Chemical Company, Milwaukee, Wis., n=5) to study the effectsof locally administered nicotine. For comparison, in some cases basicfibroblast growth factor (bFGF; 20 mcg) or Del-1 protein (0.2 M) wasadded to the pellet, rather than nicotine. Both bFGF and Del-1 are knownto induce angiogenesis. The pellet was then coated with ethylene-vinylacetate co-polymer (Elvax, Dupont, Chemcentral Corp., Chicago, Ill.)which would permit slow release of the nicotine from the pellet into thedisc. The pellet was then re-inserted into the disc before sealing thedisc with the millipore filters.

To study the systemic effects of nicotine, in some cases the animalswere administered nicotine in their drinking water (see below).

Implantation of the disc. The mice were anesthetized with 4%chloralhydrate [intraperitoneal administration (i.p.), 0.1 cc/10 g bodyweight]. The flanks and posterior surface of the thorax were shaved andcleaned with saturated 70% isopropyl alcohol. A 2-cm incision was madein the skin of the flank contra-lateral to the implantation site. Bluntdissection through the subcutaneous tissue produced a channel into whichthe saline moistened disc was inserted. The skin was closed with 5.0silk suture (Kowalski et al. (1992) Exp Mol Pathol 56(1):1-19; Fajardoet al. (1998) Lab Invest 58:718-7244).

Disc removal and preparation. Two weeks after disc implantation, themice were sacrificed with an overdose of 4% chloral hydrate (i.p.) andcervical dislocation. A careful incision was made next to the skinoverlying the implanted disc, and the disc was gently removed from theimplantation site. Attached tissue was carefully detached from the disc.After removing the disc, one filter was separated from the disc. Discswere then fixed in 10% formalin and embedded flat in paraffin.Subsequently, 5 μM sections were made in a plane through the center ofthe disc and parallel to the disc surface.

Quantitation of results. The disc sections were stained with H&E forlight microscopy and histomorphometric measurement of radial growth, andstained with toluidine blue for quantitative determination of total areaof fibrovascular growth. Using a video microscope and a computerassisted digital image analysis system (NIH Image 1.59b9), the entirearea of fibrovascular growth in the toluidine blue stained disc wascalculated and expressed in mm². As described in a previous study, totalfibrovascular growth area is directly proportional to the total area ofthe disc occupied by blood vessels (Kowalski et al. (1992) Exp MolPathol 56(1):1-19; Fajardo et al. (1998) Lab Invest 58:718-7244).Therefore, the measurement of such total area is used as an index ofangiogenesis (Kowalski et al. (1992), supra; Fajardo et al. (1998),supra).

Vascular continuity assessment. To visualize the microvessels in thedisc sections and to establish that there was continuity between thesystemic and disc vasculatures, luconyl blue dye was injected into theleft carotid artery prior to euthanizing the mice. Animals wereanesthetized using 4% chloralhydrate (i.p., 0.1 cc/10 g body weight). Anincision was made in the ventral midline of the neck. After the carotidsheath was exposed, the left carotid artery was separated from theneurovascular bundle and secured by two 4.0 silk sutures. An incisionwas made in the carotid and a 15-cm length of PE10 tubing (BecktonDickinson, Sparks, Md.) was introduced into the carotid artery andadvanced to the ascending aorta just distal to the aortic valve. About1.0 ml of luconyl blue was then slowly injected from a 1 ml syringethrough the tubing into the thoracic aorta. The presence of blue dye inthe fibrovascular network in the disc was detected by light microscopy.Microscopy revealed microvessels lined by a single layer of endotheliumand erythrocytes contained within their lumen. Luconyl blue dye wasobserved throughout the vessels of the disc.

Murine Ischemic Hindlimb Model of Peripheral Arterial Disease (PAD).Mice were anesthetized with 4% chloral hydrate [intraperitonealadministration (i.p.), 0.1 cc/10 g body weight). The medial surface ofboth hindlimbs were shaved and then cleaned with betadine solution. A1.5-cm longitudinal incision was performed, extending from the knee tothe inguinal ligament. Through this incision, the superficial femoralartery was dissected free along its length. After the distal ends ofboth the external iliac and superficial femoral arteries were ligatedwith 7.0 (Ethicon), complete excision of the femoral artery wasperformed. An additional set of mice underwent sham operation. Theincisions were then closed with discontinuous stitches of 5.0 silksuture (Ethicon). Ampicillin (1 mg/10 gm body weight) intraperitonealinjection was administered after surgical procedure.

Histological Studies

Tissue Preparation. Three weeks after surgery, mice were euthanized withan overdose of 4% chloral hydrate (i.p) and cervical dislocation, andthe adductor and semimembranous muscles were collected for capillarydensity assessment. Briefly, a longitudinal incision in the medial thighwas made to expose the entire hindlimb muscle. The adductor andsemimembranous muscles were removed and immediately frozen in OCT.Subsequently, sections 5 μm were taken from the mid-region of eachmuscle in a transverse orientation. The sections were air dried andfixed in acetone.

Capillary Densitometry. Immunohistochemistry was performed using analkaline phosphatase assay to identify the endothelial cells. An eosincounterstain was used to differentiate myocytes. Capillaries andmyocytes were identified and counted using light microscopy (20×). Foreach section, four different fields were selected and the total numberof capillaries and myocytes per field determined. These values wereaveraged to provide a determination of capillary density for eachexperimental limb. To ensure that value for capillary density was notoverestimated due to muscle atrophy, or underestimated due tointerstitial edema, capillary density was expressed as a ratio ofcapillaries to myocytes present in the same field.

Data Analysis

All data are given as mean±SEM. Statistical significance was testedusing unpaired, two-tailed t-test for comparisons between groups.Statistical significance was accepted for p<0.05.

Example 1 Systemic Effect of Nicotine Upon Angiogenesis in vivo

To study the effects of nicotine in systemically treated mice, nicotine(60 mcg/ml, n=5) was diluted in the drinking water. The mechanism ofnicotine-induced angiogenesis was studied by giving oral supplementationof indomethacin (20 mcg/ml; Sigma, n=5) and/or LNNA (6 mg/ml, Sigma,n=5) to mice having an implanted DAS both locally treated (nicotineinside the DAS) and systemically treated (nicotine diluted in drinkingwater). Concentrations of nicotine, indomethacin, and LNNA weredetermined in accordance to studies using oral supplementation of theseagents in murine models (Maxwell et al. (1998) Circulation 199898(4):369-374; Fulton et al. (1980) Int J Cancer 26(5):669-73; Rowell etal. (1983) J Pharmacological Methods 9:249-261).

Under basal conditions (untreated water, vehicle-treated disc),fibrovascular growth into the disc occurred. Vessels could be seengrowing into the disc. These vessels were in continuity with thesystemic circulation as manifested by the influx of leuconyl dye intothe disc vasculature, after systemic administration of the dye. The areaof the fibrovascular growth into the disc under basal conditions wassomewhat greater than 10 mm² (FIG. 2). With systemic administration ofnicotine, there was a dramatic increase in fibrovascular growth with anarea of 35 mm² (FIG. 2). The effect of nicotine was blocked by the NOsynthase inhibitor L, nitro-arginine (LNNA) as well as by indomethacin,indicating that synthesis of both nitric oxide and prostacyclin wererequired for the angiogenic effect of nicotine.

Example 2 Local Effect of Nicotine Upon Angiogenesis in vivo

In order to determine if local administration of L-arginine could beeffective at inducing angiogenesis, in some animals, nicotine was placedwithin a pellet that was inserted into the disc angiogenesis system(described above). When nicotine was placed in the disc (rather thanadministered in the water of the animals as described in Example 1) asimilar effect was observed. The fibrovascular growth under basalconditions (about 10 mm²) was increased to about 20 mm² (FIG. 3). Again,indomethacin or LNNA blocked the effects of nicotine. These studiesindicate that systemic or local administration of nicotine inducesangiogenesis.

Example 3 Comparison of Effects of Nicotine With Other Angiogenic Agents

The effects of nicotine were compared with the angiogenic agents bFGFand Del1. The comparison with bFGF is particularly important becausethis agent is already in clinical trials in humans for therapeuticangiogenesis. In comparison to vehicle, bFGF, Del1, and nicotine eachincreased angiogenesis to the same degree. Systemically administeredenhance angiogenesis to a much greater degree than locally administeredbFGF and Del-1 (FIG. 4). Paradoxically, the effect of systemic nicotineadministration was greater than local nicotine administration, eventhough systemic nicotine administration undoubtedly produced lower locallevels in the disc. The paradox may be explained by the experimentsshown below where it was observed that intermediate doses of nicotineadministered intramuscularly have the greatest effect. At higherintramuscular doses of nicotine, less angiogenesis is observed.

Example 4 Induction of Angiogenesis in the Murine Hindlimb IschemiaModel of Peripheral Arterial Disease

To provide more compelling evidence for the therapeutic angiogeniceffects of nicotine, the angiogenic effects of nicotine were examined ina model of arterial occlusive disease, the murine ischemic hindlimb(described above). Daily intramuscular injections of nicotine solutionor vehicle were administered (50 μl ) for a period of three weeks. Fivegroups of animals received 0, 3, 30, 300 or 3200 ng/kg of nicotine byintramuscular injection daily (represented in FIGS. 6 and 7 as 1×(0.0811 ng nicotine in 50 μl saline (=0.003 μg/kg), 10× (0.811 ngnicotine in 50 μl saline (=0.03 μg/kg), 100× (8.11 ng nicotine in 50 μlsaline (=0.3 μg/kg), and 1000× (8.11 ng nicotine in 50 μl saline (=3.2μg/kg)). As shown in FIG. 5, 3 weeks after surgery, capillary density(capillaries/myocyte) was increased in operated limbs (ischemic) incomparison to non-operated limbs (non-ischemic) consistent with a basalangiogenic response to ischemia.

As shown in FIGS. 6 and 7, nicotine enhanced the angiogenic response toischemia relative to controls. With vehicle control, 0.35capillaries/myocyte were detected in the ischemic limb. At anintermediate dose of 0.03 μg/kg, nicotine nearly doubled angiogenicresponse (to 0.67 capillaries/myocyte). At the highest dose of nicotine,angiogenesis was not increased; indeed at this dose some toxicity wasobserved, with evidence of interstitial edema and myocyte necrosis. Theangiogenic effect remained local to the site of nicotine injection,since no angiogenic effect was detected in a non-ischemic hindlimbs(FIG. 6). Thus, local intramuscular administration of these doses ofnicotine did not result in a systemic angiogenic effect.

These studies indicate that nicotine enhances angiogenesis in a murinemodel of human peripheral arterial disease, and that nicotine receptoragonists are useful for therapeutic angiogenesis.

Example 5 Effect of nAChR Agonists, VEGF, and FGF on Endothelial CellMigration

FIG. 8 shows the results of an experiment that further tests theinteraction between the pathways mediated by the VEGF, FGF and nAChReceptors.

In this experiment, the effects of VEGF, FGF and nicotine in anendothelial cell (EC) migration assay were observed. The EC migrationassay involves growing a confluent endothelial monolayer, and thenscraping away half of the cells. The number of endothelial cells thatmigrate over time into the denuded region was observed. Unstimulatedcells will migrate into the region. Angiogenic agents will acceleratethis migration.

In this histogram shown in FIG. 8, the number of migrating cells isexpressed as a percentage of the number of endothelial cells migratinginto the region under unstimulated conditions. VEGF (1 ng/ml; VEGF(1);hatched bar) increases EC migration to the same degree as FGF orNicotine; high dose VEGF (10 ng/ml; VEGF(10)) gives the greatestresponse.

Intriguingly, neostigmine (Neo; 10⁻⁶ M) alone increased EC migrationover 50%, giving a similar effect as nicotine (Nic), FGF, or low dose (1ng/ml) VEGF. The combination of neostigmine and low dose (1 ng/ml) VEGF(second hatched bar; VEGF(1)+neo(10⁻⁶ M)) gave a greater response thanhigh dose VEGF.

Neostigmine blocks acetylcholinesterase, the enzyme that degradesacetylcholine. Endothelial cells contain acetylcholinesterase on theirplasmalemma.

This experiment indicates that acetylcholine is produced by endothelialcells, and stimulates migration. Neostigmine reduces the breakdown ofacetylcholine, and increases the effect of acetylcholine. Acetylcholinethen stimulates nACh receptors on the endothelium to acceleratemigration. Notably, neostigmine (by increasing the bioavailability ofacetylcholine) augments the effect of low dose VEGF. This study showsthat endogenous VEGF and acetylcholine work together to accelerateangiogenic processes.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A method of stimulating therapeutic angiogenesis in a mammal having acondition that is treatable by stimulating angiogenesis, the methodcomprising administering to the mammal a nicotine receptor agonist, or apharmaceutically acceptable salt thereof, in an amount effective tostimulate angiogenesis, wherein said administration results instimulation of therapeutic angiogenesis in the mammal, and wherein saidadministering is by topical administration.
 2. The method of claim 1,wherein the nicotine receptor agonist is nicotine, cotinine,norcotinine, nornicotinine, nicotine N-oxide, cotinine N-oxide,2-hydroxycotinine, or 5-hydroxycotinine, or a pharmaceuticallyacceptable salt thereof.
 3. The method of claim 2, wherein the nicotinereceptor agonist is S-3-(1-methyl-2-pyrrolidinyl)pyridine, or apharmaceutically acceptable salt thereof.
 4. The method of claim 1,wherein said administering is at a site of a surgical wound, in oraround a wound, in or around a skin graft, or in or around an ulcer. 5.The method of claim 1, wherein the nicotine receptor agonist is in asemi-solid formulation.
 6. The method of claim 5, wherein the semi-solidformulation is a gel.
 7. The method of claim 1, wherein the nicotinereceptor agonist is formulated with carboxymethylcellulose.
 8. Themethod of claim 7, wherein the carboxymethylcellulose is sodiumcarboxymethylcellulose.
 9. The method of claim 7, wherein the nicotinereceptor agonist is formulated for sustained release.
 10. The method ofclaim 1, wherein the nicotine receptor agonist is administered in anamount of about 0.001 mg to 1 mg per dose.
 11. The method of claim 1,wherein the mammal is a human.
 12. The method of claim 1, furthercomprising administering an agent selected from vascular endothelialgrowth factor, fibroblast growth factor (FGF), acidic FGF, and basicFGF.
 13. A method of stimulating therapeutic angiogenesis in a mammalhaving a condition that is treatable by stimulating angiogenesis, themethod comprising administering to the mammal a nicotine receptoragonist, or a pharmaceutically acceptable salt thereof, in an amount ofabout 0.001 mg to 1 mg per dose, wherein said administration results instimulation of therapeutic angiogenesis in the mammal.
 14. The method ofclaim 13, wherein the nicotine receptor agonist is nicotine, cotinine,norcotinine, nornicotinine, nicotine N-oxide, cotinine N-oxide,2-hydroxycotinine, or 5-hydroxycotinine, or a pharmaceuticallyacceptable salt thereof.
 15. The method of claim 13, wherein the mammalis a human.
 16. The method of claim 13, further comprising administeringan agent selected from vascular endothelial growth factor, fibroblastgrowth factor (FGF), acidic FGF, and basic FGF.
 17. The method of claim13, wherein the nicotine receptor agonist isS-3-(1-methyl-2-pyrrolidinyl)pyridine, or a pharmaceutically acceptablesalt thereof.